Osteoclasts, which differentiate from monocyte/macrophage lineage, play a critical role in bone metastasis. Incomplete phagocytosis leads to the formation of osteoclast-tumor hybrid cells (OTHCs), which co-expressing bone-resorbing markers (TRAP, cathepsin K (Cat K)) and epithelial/oncogenic markers (cytokeratins, pSTAT3/JAK1). These hybrid cells enhanced bone resorption capacity and oncogenic properties. Understanding the detail mechanism of OTHCs formation provides potential therapeutic target. In this study, we observed large population of OTHCs in tissue samples of bone metastatic patient. These OTHCs engulfed tumor cells and formed multinucleated giant cells exhibiting oncogenic features, including pSTAT3, JAK1, and Ki67 expression. Their formation may also be associated with aging. In addition, preliminary results from co-culture experiments and a bone metastasis mouse model demonstrated a decline in oxidative phosphorylation (OXPHOS) in both aging mouse tissues and bone marrow–derived macrophages (BMDMs) from aged mice. This impairment may increase the risk of incomplete phagocytosis, thereby promoting the formation of OTHCs. Furthermore, inhibition of OXPHOS on Raw 264.7 cells with STAT6 inhibition facilitated hybrid cells formation. Taken together, this study highlights that OXPHOS dysfunction promotes the formation of OTHCs in bone metastasis.
Other authors: N/A
Understanding how the immune system “recognizes” foreign bodies, such as nanocarriers shuttling anti-cancer drugs, engineered CAR-T cells, and cancer cells themselves, is essential to designing effective cancer treatments. The innate immune system depends on the complement cascade, a 500-million-year-old protein network which deposits C3b on protein-binding sites on foreign body surfaces, labeling them for phagocytic clearance. Unwanted immunogenicity prevents therapies from reaching their targets and can lead to adverse patient reactions. However, the principles governing how the system “decides” to coat an NC and how this alters immune uptake have eluded researchers. We utilize a multi-scale systems approach to investigate this question. We integrate ordinary differential equations and an agent-based model, validated by experiment, to reveal that complement activation follows a percolation phase transition governed by NC surface protein density. We reveal that a critical percolation threshold separates uncoated and C3b-coated NC regimes; NCs above the threshold exhibit multistability within a bimodal distribution, occupying states of low, high, or no C3b coverage. Using our Dynamically Triangulated Monte Carlo membrane simulation model, we show that NC–membrane interactions vary across these regimes. High-C3b NCs form numerous ligand–receptor bonds dominated by C3b-mediated adhesion and induce local curvature—suggesting greater internalization propensity. Low-C3b NCs require higher site density to achieve similar effects, mediated by both C3b and other NC surface proteins. NCs lacking C3b evade immune recognition. This biophysics-based understanding of nanocarrier immunogenicity can help inform the design of safer and more effective treatments. * We acknowledge support in part from NCI
Other authors: Sahil Kulkarni, Ravi Radhakrishnan
Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy characterized by gene mutations and chromosomal abnormalities with a 5-year survival rate of only 33%, largely due to relapse driven by treatment-resistant leukemia stem cells and metabolic reprograming. Therefore, a systematic framework that allows us to characterize and predict treatment response is urgently needed. Previously, we have shown that state-transition models predict AML disease evolution based on changes of mRNA and miRNA transcriptomes, where each transcriptome is modeled as a particle undergoing Brownian motion in a double-well potential. Here we propose a two-dimensional (2D) state-transition model to predict AML response to chemotherapy using time-series peripheral blood samples collected from a mouse model of AML. Using this data, we construct a multiomic state-space, which reveals a treatment-induced alteration to the miRNA-mRNA dynamics. We use our 2D state-transition model to quantify the effects of chemotherapy on multiomic potential and find that our model accurately predicts response to treatment over time. Finally, we use the state-space critical points identified by the model as classifiers of specific disease states to guide a longitudinal genomic analysis. This analysis identifies changes in metabolic pathways such as oxidative phosphorylation, glycolysis and fatty acid metabolism over disease progression and response to chemotherapy. Our approach provides an innovative framework to understand treatment response and improve therapeutic strategies in AML.
Other authors: Ziang Chen, Yu-Hsuan Fu, David E. Frankhouser, Denis O’Meally, Sergio Branciamore, Jihyun Irizarry, Bin Zhang, Lianjun Zhang, Guido Marcucci, Ya-Huei Kuo, Russell C. Rockne
The fallopian tube (FT) is a specialized organ in the female reproductive tract key to human reproduction and ovarian cancer development. Loss of ciliated cells in the FT epithelium is one of the earliest features of the precancerous landscape. FTs experience shear stress from follicular fluid expulsion and peristaltic-ciliary movements, with cilia height, density, and beating frequency influencing fluid transport and response to mechanical cues. Although gamete and embryo transport dynamics in FT are well-established, the impact of shear stress on FT ciliary function is unexplored. This study aims to establish effects of shear stress on FT ciliated cells using mouse- and patient-derived cells in a microfluidic platform. An in vitro microfluidic system to apply tunable shear stress on FT cells was established and shear stress acting on individual cells was characterized using COMSOL. Primary epithelial cells from human FT or mouse oviduct organoids were exposed to shear stress (1 or 5 dyne/cm²) for 24 hours. Shear-induced phenotypes were evaluated using qRT-PCR and immunofluorescence for cilia markers. Cell viability was unchanged across all conditions. In human FT epithelial cells, 1 dyne/cm² shear stress upregulated cilia-related (FOXJ1, CAPS) and differentiation markers (OVGP1, SOX17), while reducing proliferation genes (CCNE1, STMN1). Mouse oviduct cells showed reduced proliferation markers and decreased or unchanged differentiation markers at both shear levels. Acetylated tubulin staining confirmed increased ciliary proteins in human but not mouse cells under shear stress. Overall, shear stress promoted differentiation in human FT epithelium, but mouse cells showed reduced ciliation, indicating species-specific responses to flow.
Other authors: Geeta Mehta
About 70% of pancreatic ductal adenocarcinoma (PDAC) patients present with metastatic disease at diagnosis, with the liver and lung among the most common sites of distal metastasis. Notably, liver metastases are associated with poorer patient outcomes and diminished responsiveness to systemic therapy compared to isolated metastases in other organs. These clinical differences suggest that organ-specific features of the tumor microenvironment (TME) may influence disease progression and therapeutic efficacy. Emerging evidence indicates that the immune landscape differs significantly between primary PDAC and its metastatic sites. Conventional dendritic cells (cDCs) are critical for mounting anti-tumor T cell responses. We have previously reported that cDCs are scarce and dysfunctional in the primary PDAC TME, however, the infiltration and function cDCs at metastatic sites remain poorly understood. Our recent data suggests that the site of metastasis impacts cDC abundance and localization, with liver metastases showing distinct patterns of cDC organization compared to both primary tumors and lung metastases. The impact of these differences on T cell recruitment and activation in metastatic PDAC remains unclear.
Other authors: Alyssa G. Weinstein, Yu-Lan Kao, Blake Sells, Brett Knolhoff, Li Ding, Paul M. Grangenett, Liang-I Kang, David G. DeNardo
Late-stage epithelial ovarian cancer (EOC) has high recurrence rates and poor prognosis due to residual microscopic metastases remaining after surgical tumor removal. While standard chemotherapy can reduce metastatic deposits, lack of tumor specificity and high toxicity limit treatment success. Our group previously demonstrated a fluorescence microendoscope capable of imaging metastatic tumors using various fluorescent markers. Combined with tumor-targeted photodynamic therapy (PDT) and laser ablation, this approach offers a promising alternative for treating recurrent EOC with improved outcomes. Multiplexed multiphoton microendoscopy requires innovations in laser pulse engineering, optical design, and hardware for real-time tumor imaging in vivo. Conventional detector technology such as photomultiplier tubes (PMTs) are expensive, bulky, and have limited detection range for this application. Silicon photomultipliers (SiPMs) represent an emerging detection technology offering significant advantages while maintaining single-photon sensitivity and rapid response time: lower cost, scalability, damage resilience, and higher dynamic range. This work presents a prospective framework for incorporating a multispectral SiPM array detector into our microendoscope system to obtain fluorescent images of metastases at 20 frames/sec. We will demonstrate the feasibility of SiPM-based detection for real-time imaging and evaluate performance compared to conventional PMT systems. This advancement will enable more accessible and robust microendoscopic platforms for targeted EOC treatment.
Other authors: Liam Price, Kai Zhang, Gunar Schirner, Michael Giacomelli, Bryan Spring
Multiple myeloma (MM) is an incurable B-cell malignancy for which T-cell engaging antibodies (TCEs) targeting B-cell maturation antigen (BCMA) have shown promising clinical activity. However, resistance to TCEs remains a major challenge. Two key mechanisms—antigen loss and immune suppression—are implicated in treatment failure, yet their relative contributions and potential synergy are not fully understood. To address this gap, we developed a data-informed agent-based model (ABM) that simulates the MM tumor microenvironment (TME) under TCE therapy, incorporating experimentally calibrated dynamics of antigen downregulation and immune dysfunction. Simulations revealed that while neither antigen loss nor immune suppression alone is sufficient to drive resistance, their combination leads to markedly diminished TCE efficacy and tumor persistence. These findings suggest that immune escape in MM is a multifactorial process requiring multi-pronged therapeutic strategies. Our study underscores the importance of preserving antigen expression and restoring immune activity to overcome TCE resistance and improve patient outcomes.
Other authors: Karl Nyman, Ryan T. Bishop, Kenneth Shain, Conor Lynch, David Basanta
Advances in multiregion sequencing have revealed extensive intratumor heterogeneity (ITH) — the presence of genetically distinct subclones within a single tumor. These findings support a branching evolution process (BEP), in which multiple subclonal lineages evolve in parallel from a common ancestor. ITH profoundly influences tumor behavior, including hallmarks of cancer, and growth and invasion phenotypes. It also underlies the emergence of therapeutic resistance, as distinct subpopulations may harbor mutations that confer survival advantages. We have built a 2D, multi-scale, data-driven agent-based model (ABM) for BEP with Hallmark Integration (BEP-HI) that simulates melanoma evolution under the hallmarks of uncontrolled proliferation, resistance to apoptosis, immune evasion, and genetic instability. The model is calibrated for BRAF-associated superficially spreading melanoma using growth rates data. We find three ITH regimes: clonal sweep where a single dominant clone overtakes the tumor population, subclonal sweep where multiple subclones cluster with their own, and fractal with high heterogeneity and little spatial clustering. We use cross-PCF to quantify spatial relations and find that the most aggressive clone (highest count of driver gene mutations) closely clusters with the second most aggressive. Additionally, we observe that ICs are much more present in hot tumors than cold, and show opposing dynamics as base mutation rates are in increased in each case.
Other authors: Trachette Jackson
Super-resolution microscopy is widely used to quantify the spatial distribution of histone modifications. However, many features of nucleosome organization exist close to or below the resolution limit of super-resolution, and measurements made directly from super-resolution images are prone to biases from repeated blinking of individual fluorophores and the length of the probe used. To address these limitations, we have developed a simulated ground truth chromatin model upon which we simulate probe binding, fluorophore blinking dynamics, noise, and image reconstruction. This model can be used to investigate the effects of biases and to validate histone quantification techniques. Super-resolution microscopy has enabled studies that probe protein spatial distribution at the nanoscale. This, in turn, has made it possible to study the distribution of post-translational modifications in the nucleus. Cancer is associated with widespread alterations in gene expression. It is of interest to classify the resulting change in the spatial distribution of post-translational modifications. However, there is a lack of studies that examine the interactions of multiple histone modifications in a single nucleus. We quantified the individual distribution and clustering behaviors of H3K27me3 and H3K27ac and classified the level of contact between these histone modifications using spectroscopic single-molecule localization microscopy (sSMLM). We also associated the detected changes in these parameters with degrees of cancer malignancy and with drug-induced perturbations in methylation machinery.
Other authors: Benjamin Brenner, Marcelo Carignano, Luay Almassalha, Daniela Matei, Vadim Backman, Igal Szleifer
Single-molecule localization microscopy (SMLM) achieves super-resolution by analyzing individual fluorescence emissions. Spectroscopic SMLM (sSMLM) extends this by capturing spectral data but suffers from reduced precision due to photon splitting. We present a symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM system that maximizes photon use for both spatial and spectral analyses. Fluorescence photons are equally split and symmetrically dispersed using two identical dual-wedge prisms, with spatial positions and spectra computationally extracted using all collected photons. This approach improves spatial and spectral precisions by 27% and 48%, respectively, over prior sSMLM systems. Using a single excitation laser, we achieve multiplexed imaging of peroxisomes, microtubules, and mitochondria labeled with spectrally overlapping dyes (DY-634, AF647, CF660C). We also demonstrate massively parallel tracking of spectrally tagged nanoparticles at concentrations five times higher than previously reported. The entire system is integrated into a compact module, facilitating seamless adoption into existing SMLM setups.
Other authors: Wei-Hong Yeo, Benjamin Brenner, Youngseop Lee, Junghun Kweon, Cheng Sun, Hao F. Zhang
Triple-negative breast cancer (TNBC) comprises 15–20% of cases and remains the most clinically challenging subtype due to the lack of targeted therapies and high heterogeneity. Previous studies have focused on defining molecular subtypes of TNBC to better understand the molecular heterogeneity and guide treatment. However, the metabolic heterogeneity and its impact on clonal interactions and tumor progression is less understood. To investigate this, we generated single-cell-derived clonal populations from the MDA-MB-231 TNBC cell line and cultured these clones under high (4.5 g/L) and low (1 g/L) glucose conditions. Transcriptomic profiling revealed that clones separated into two clusters distinguished by ESAM expression. Each clone exhibits distinct metabolic specialization based on the ESAM expression showing complementary metabolic profiles between clones. We then assessed clonal interactions by growing populations in monoculture and coculture. Cocultures pairing ESAM+ and ESAM– clones exhibited cooperative interactions that significantly enhanced growth compared to monocultures. These findings indicate that metabolic specialization drives nutrient-dependent cooperation between clonal populations. Our study highlights the importance of metabolic heterogeneity and cooperation in TNBC progression and suggest that disrupting nutrient-dependent metabolic interactions may be a novel therapeutic approach to target tumor heterogeneity and inhibit tumor growth.
Other authors: Amy Brock
Lung adenocarcinoma (LUAD) is a leading cause of cancer-related deaths, primarily due to tumor heterogeneity and drug resistance. The tumor microenvironment (TME) consists of tumor cells, stromal cells, and immune cells. These distinct cellular neighborhoods respond differently to therapeutic agents based on their spatial organization and composition. However, effective in vitro or ex vivo models that preserve the native spatial organization of the TME for drug testing are lacking. To address this, we are developing an in vitro co-culture system that integrates cancer cells, tumor-associated fibroblasts, and immune cells. Additionally, we are creating an ex vivo culture system using precision-cut lung slices (PCLS) from freshly resected LUAD tissues. We will employ advanced multimodal spatial-omic technologies (Xenium and Phenocycler Fusion) to characterize the TME's distinct cellular compartments and their transcriptional states. Our recent computational framework will integrate spatially resolved multi-omic data to identify functional gradients within the TME. This research aims to enhance personalized treatment strategies and improve patient outcomes by elucidating the spatial dynamics of tumor-stroma-immune interactions and their impact on drug efficacy within the TME.
Other authors: Dina Hany, Anum Khan, Jacob Chang, Sylvia K. Plevritis
Photodynamic therapy (PDT) research benefits from accessible, stable, and automated light delivery systems for high-throughput in vitro studies. A low-cost, easy-to-use setup compatible with standard multiwell plates would enhance the scalability and reproducibility of PDT experiments. We present a custom LED array platform designed for cell culture PDT, offering programmable, wavelength-specific light delivery across multiple wells. The system includes a water-cooling loop to maintain a constant operating temperature, ensuring stable optical output in both intensity and spectrum over time. Two custom actuator arms, combined with pulse-width modulation control, enable automated and programmable light exposures, supporting diverse experimental protocols. The system operates at approximately 690 nm and delivers an adjustable irradiance up to 400 mW per square centimeter. The modular design allows straightforward adaptation to other LED wavelengths to accommodate various photosensitizers. Compared with previously reported LED-based PDT setups, this system offers improved spectral and power stability. The light dose output was validated by comparison with traditional laser-based PDT, confirming its accuracy and reliability. This open-source illumination platform provides a practical and scalable solution for photomedicine research. Its automated operation, spectral flexibility, and validated performance make it a valuable tool for researchers developing and testing PDT treatments across a range of cell models.
Other authors: Sudip Timilsina, Matthew Waguespack, Eric M. Kercher and Bryan Q. Spring
Background:
Intratumoral hypoxia is a hallmark of solid tumours that drives both epithelial–mesenchymal transition (EMT) and cell cycle alterations. Our lab has previously shown that intratumoral hypoxia induces partial/hybrid EMT. However, the mechanisms that maintain epithelial features under hypoxic stress remain poorly understood. A downstream target of hypoxia and a mediator of cell cycle arrest, p27, is shown to be stabilized under stress conditions via p53-dependent mechanisms. The p53–MDM2 axis can also stabilize OVOL2, a known suppressor of full EMT. However, the spatiotemporal dynamics of these molecular players within hypoxic tumour microenvironments remains unclear, which is the focus of this study.
Methods & Results:
Using a three-dimensional polyethylene glycol-based microtumour model that generates intrinsic hypoxic gradients, we performed a spatiotemporal analysis of cell cycle regulators and partial EMT markers. Temporal profiling via qRT-PCR, western blotting and immunofluorescence revealed a stable partial EMT phenotype, with sustained expression of E-cadherin and Vimentin. Cyclin D1 decreased over time, accompanied by increase in p27 levels suggesting G1 phase arrest. The expression of GRHL2 and OVOL2 was maintained through 72h, and reduced thereafter under prolonged hypoxia without loss of E-Cadherin.
Conclusion:
Our data suggests a model in which hypoxia induces G1 cell cycle modulation alongside partial EMT, in breast cancer cells. The enrichment of p27 and OVOL2 points to a possible regulatory link, between cell cycle arrest and partial EMT under hypoxic stress. These findings offer new insights into the coordination of EMT and cell cycle regulation in 3D tumor models.
Other authors: N/A